CN110140810B

CN110140810B - Preparation method of fermented yellow wine lees for feed and fermented yellow wine lees

Info

Publication number: CN110140810B
Application number: CN201910436143.XA
Authority: CN
Inventors: 汪以真; 王成; 苏维发; 姜子鹏; 郝丽红; 张煜; 路则庆; 王凤芹
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-05-23
Filing date: 2019-05-23
Publication date: 2022-03-15
Anticipated expiration: 2039-05-23
Also published as: CN110140810A

Abstract

The invention discloses a preparation method of fermented yellow wine lees for feed and the fermented yellow wine lees. 1) Preparing a fermentation raw material; 2) preparing a culture medium; activating the fermented seed liquid, namely mixing the bacillus subtilis: mixing and inoculating pediococcus pentosaceus according to the ratio of 1: 1-1: 1.5; 3) packaging and fermenting for 3-4 days. The invention can obviously reduce the content of resistant starch and fiber in the yellow wine lees, improve the content of crude protein, small peptide, probiotics and organic acid, improve the pig bionic digestibility of the yellow wine lees and has better application prospect.

Description

Preparation method of fermented yellow wine lees for feed and fermented yellow wine lees

Technical Field

The invention belongs to the technical field of animal feed, and particularly relates to a preparation method of fermented yellow wine lees for feed and fermented yellow wine lees.

Background

Yellow wine belongs to brewed wine, is a special product of China, and occupies a place in three brewed wines (yellow wine, wine and beer) in the world. In recent years, yellow wine industry in China has been greatly developed, more than 700 yellow wine production enterprises exist, the yellow wine yield in 2014 nationwide is 273 ten thousand tons, and the yellow wine yield in Zhejiang area accounts for more than half of the national yield (China industry information network, 2015). The vinasse yield of the yellow wine is about 20-30 percent, and the amount is considerable. Yellow wine lees are byproducts in yellow wine brewing, and compared with white wine lees and beer lees, the yellow wine lees have lower fat content and richer contents of crude protein, starch, lysine, methionine and threonine. In addition, the proportion of lysine, methionine and threonine in the yellow wine lees in the total amino acids respectively reaches 3.61%, 2.86% and 4.05%, which are higher than 3.11%, 0.62% and 1.77% of the soybean meal. However, the yellow wine lees is not obviously improved in animal growth performance when being used for feeding the growing pigs by 12 percent instead of bean pulp. In addition, the apparent digestibility of the pig to the feed crude protein in the whole intestinal tract is reduced from 82.73% to 78.60%, and the digestibility of the pig to lysine, methionine and threonine is reduced from 79.72%, 73.52% and 74.74% to 74.75%, 69.25% and 68.99%, respectively. This indicates that the animal has low digestion and absorption capacity to the yellow wine lees protein, and the feeding effect of the yellow wine lees is influenced.

There are many factors that affect the digestion and utilization of yellow wine lees, and anti-nutritional factors are one of the reasons. 30% of yellow wine lees dry matter is starch, and 48-50% of the starch is resistant starch. Although the intake of proper amount of resistant starch is beneficial to regulating the intestinal microbial flora, the excessive resistant starch can stimulate the intestinal tract to wriggle, reduce the retention time of the feed in the intestinal tract and cause the livestock to loose stool and even diarrhea. In addition, the resistant starch has a large molecular structure, has a physical embedding effect and a barrier effect on other nutrient substances, and influences the digestion and absorption of animals on feed nutrients. Meanwhile, resistant starch in the yellow wine lees can form an agglutination complex with fiber, protein and the like, and the digestibility is further reduced.

Although the research of fermenting the yellow wine lees is available at present, the Candida tropicalis fermented yellow wine lees used by Zhao Jian nations and the like has the advantages that the crude protein is 57.27 percent, the true protein is 50.64 percent, the starch degradation rate is 58.99 percent, and the feeding value of the yellow wine lees is effectively improved. The Candida tropicalis, the Trichoderma viride and the Aspergillus oryzae are used for fermenting yellow wine lees by the Wangjiangjun and the like, the content of true protein of a pilot-scale fermentation product is increased by 3.37 percent by detection, the content of starch is reduced by 5.58 percent by detection, and the egg yield and the egg weight can be obviously improved by feeding laying hens with 10 percent of the fermented yellow wine lees instead of protein feed. Therefore, in the prior art, mould fermentation is mainly used, hypha can be generated to destroy the sensory characteristics of the fermented yellow wine lees, the application research of the fermented yellow wine lees by using bacteria in live pig breeding is less at present, and the specific application effect is not clear. Therefore, attention should be paid to the development and utilization of yellow wine lees for resisting the degradation of nutritional factors such as resistant starch and fiber and improving the digestibility of the yellow wine lees.

The method improves the nutritive value of the feed by means of microbial fermentation, and the production of green and efficient feed becomes a hotspot of research in the breeding industry under the background of 'no resistance'. The microbial fermented feed can provide beneficial bacteria and metabolites thereof, regulate the pH value of gastrointestinal tracts, maintain the balance of intestinal flora, reduce the use of antibiotics, improve the nutrient digestibility, develop and utilize new feed resources and relieve the problem of grain safety. Meanwhile, lactic acid generated by the probiotics can inhibit the growth and reproduction of pathogenic bacteria and pathogens such as escherichia coli in the feed, and the storage time of the feed is prolonged. Yellow wine lees is a low-value feed raw material of ground source in Jiangzhe and Zhejiang areas in China, and reports on how to improve the nutritional composition of the feed raw material by utilizing the solid state fermentation of microorganisms and expanding the application value of the yellow wine lees as the characteristic protein feed raw material of the ground source in the live pig breeding are limited.

CN107535671A discloses a microbial fermentation yellow wine lees feed for improving rumen protein utilization rate and a preparation method thereof, wherein a feed substrate is prepared by crushing and mixing 40% of yellow wine lees, 50% of bran, 5% of cassava residues and 5% of glucose according to weight percentage, performing solid state fermentation by mixing bacillus subtilis KF-2 and candida utilis KF-1, and drying. However, the yellow wine lees of the invention has a low proportion, belongs to mixed feed of bran, yellow wine lees and cassava residues, and adopts the compatible fermentation of bacillus subtilis and yeast, mainly aiming at a fermentation product for improving the utilization rate of rumen protein of cattle.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to solve the technical problem of providing a feed fermented yellow wine lees

The preparation method and the fermented yellow wine lees thereof.

A preparation method of fermented yellow wine lees for feeding comprises the following steps:

1) preparing fermentation raw materials

Premixing the yellow wine lees and the bran raw materials according to the mass ratio of 8: 2-9: 1;

2) activating and fermenting seed liquid and inoculating

Inoculating bacillus subtilis CW4 to the LB liquid culture medium at 37 ℃ for culturing for 10-12 h to obtain bacillus subtilis fermentation seed liquid, inoculating pediococcus pentosaceus CWPP to the MRS liquid culture medium at 37 ℃ for culturing for 16-18 h to obtain pediococcus pentosaceus fermentation seed liquid. Uniformly mixing the two fermentation seed liquids with water according to a proportion (bacillus subtilis: pediococcus pentosaceus =1: 1-1: 1.5) to obtain a mixed activated fermentation seed liquid, inoculating the fermentation raw material prepared in the step 1) to enable the total water content to be 40% -45% of the total mass of the fermentation system, and uniformly stirring; pediococcus pentosaceus (f) mentioned abovePediococcus pentosaceus) Purchased from China general microbiological culture Collection center, and the catalog number of the strains is as follows: 1.300, CWPP is our name; bacillus subtilis (A), (B) and (C)Bacillus subtilis subsp. subtilis) CW4 is also an existing strain and is disclosed in CN106167782A of the present applicant for the first time, wherein the accession number of the common microorganism center of the chinese committee for culture collection of microorganisms is cgmccno: 12825 of Largehead tobacco leaf;

3) fermentation of

Putting the fermentation system into a fermentation device, and fermenting at room temperature for 3-4 days.

Fermented yellow wine lees obtained by the preparation method of the fermented feed.

Compared with the prior art, the invention has the following beneficial effects:

the compatible strain is utilized, so that the content of resistant starch and cellulose in yellow wine lees is obviously reduced, the content of probiotics, crude protein, small peptide, digestive enzyme and organic acid is increased, the structure of amino acid is improved, and the bionic digestibility of piglets on the yellow wine lees is increased.

Drawings

FIG. 1 is a scanning electron micrograph of yellow wine lees before and after fermentation.

Detailed Description

The invention is further illustrated by the following examples.

Example 1 preparation method of yellow wine lees fermented in laboratory

20 g of substrate (80% yellow wine lees and 20% wheat bran) was inoculated with 3% (v/w) (1.0X 10)⁸ CFUMl) of Bacillus subtilis CW 4. The substrate was fermented in a 100 mL Erlenmeyer flask, covered with a sterile membrane (aerobic conditions), and sterile water was added on a dry basis to achieve a water content of 40%. The substrate was fermented at 37 ℃ for 36 h. First stage post fermentation, mixed fermentation inoculated with 3% (v/w) (1.0X 10)⁸CFU/ml), plugging a rubber plug at the bottle mouth (anaerobic condition), fermenting for 48 h at 37 ℃ under the anaerobic condition, taking part of wet sample, and rapidly detecting microbial metabolites and in-vitro digestibility. The remaining samples were treated at 105 ℃ for 20min to terminate the fermentation. The samples were then dried at 60 ℃ for 36h, cooled and ground for nutritional analysis.

TABLE 1 nutritional component Change before and after fermentation of yellow wine lees

Project%	Yellow wine lees	Fermented yellow wine lees
			Dried substance	88.06±1.03	89.21±1.78
Lignin	1.75±1.56^a	1.28±1.60^b
			Cellulose, process for producing the same, and process for producing the same	5.30±0.50^a	3.42±0.27^b
Acid detergent fiber	11.86±1.38	8.78±1.50
			Acid detergent fiber	20.19±1.62^a	16.27±0.85^b
Total dietary fiber	11.4±41.37	13.35±0.52
			Total starch	16.14±2.36^a	11.94±0.65^b
Amylose starch	11.87±1.62^a	9.00±0.46^b
			Amylose starch	4.27±0.77^a	2.95±0.20^b
Crude protein	34.05±0.46^b	37.51±0.47
			Acid soluble protein	17.32±0.64^b	22.61±0.98^a
Free amino acids	0.74±0.06^b	3.20±0.35^a
			Small peptides	16.58±0.59^b	19.42±0.63^a
Crude fat	3.06±0.09	3.46±0.17
			Ash content	4.81±0.32	3.83±0.34
Calcium carbonate	0.22±0.02	0.18±0.02
			Phosphorus (P)	0.29±0.02^a	0.17±0.05^b
Arginine	1.12±0.19	1.16±0.18
			Histidine	0.50±0.02	0.48±0.01
Isoleucine	1.20±0.02	1.20±0.06
			Leucine	1.56±0.07^b	2.30±0.17^a
Lysine	0.40±0.02^b	0.56±0.02^a
			Methionine	0.36±0.04^b	0.65±0.05^a
Phenylalanine	1.13±0.17	1.36±0.10
			Threonine	0.58±0.04^b	1.03±0.19^a
Valine	1.13±0.09^b	1.30±0.05^a
			Aspartic acid	1.25±0.06^b	1.44±0.05^a
Serine	1.16±0.16^b	1.81±0.10^a
			Glutamic acid	3.87±0.12^b	4.26±0.05^a
Glycine	1.03±0.06	1.06±0.05
			Alanine	1.34±0.11^b	2.02±0.18^a
Cysteine	0.70±0.03	0.77±0.05
			Tyrosine	1.79±0.17	1.52±0.15
Proline	1.14±0.04	1.20±0.01
			Total amino acids	20.29±0.37^b	24.13±0.84^a

As can be seen from Table 1, compared with unfermented yellow wine lees, the content of crude fiber, washing fiber, lignin and other fiber indexes in corn wine lees and yellow wine lees is reduced, the content of crude protein, acid soluble protein and total amino acid is increased, the content and pH of resistant starch are reduced, and the content of partial restrictive amino acid and total amino acid is increased.

TABLE 2 microbial and organic acid changes before and after fermentation of yellow wine lees

Item	Yellow wine lees	Fermented yellow wine lees
			Pediococcus pentosaceus,. times.10⁸ CFU/g	-	3.30
Bacillus, x10⁸ CFU/g	-	3.20
			Enterobacter,. times.10⁴ CFU/g	2.68	-
Mold,. times.10² CFU/g	3.60	-
			Total microorganisms,. times.10⁸CFU/g	-	10.25
Lactic acid mmol/Kg	14.30±1.89^b	115.21±12.1^a
			Formic acid mmol/Kg	14.43±1.14^b	22.65±2.97^a
Acetic acid mmol/Kg	17.35±1.63^b	25.27±4.24^a
			Propionic acid mmol/Kg	0.03±0.00^b	0.40±0.04^a
Butyric acid mmol/Kg	9.41±0.46	10.94±1.40
			pH	6.43±0.10^a	4.34±0.16^b

As can be seen from Table 2, the lactic acid content accumulated to 115.21mmol/Kg after the yellow wine lees was fermented for 72 hours, the acetic acid and propionic acid contents increased significantly, the feed pH decreased gradually to 4.34, and the numbers of Bacillus subtilis and Pediococcus pentosaceus reached 3.30X 10 respectively⁸And 3.20X 10⁸The total number of bacteria after fermentation reaches more than 10 hundred million, and the content of probiotics is rich. In conclusion, the obtained fermented mixed feed has low anti-nutritional factors and rich lactic acid and probiotics contents.

TABLE 3 bionic digestibility of pig before and after fermentation of yellow wine lees

Project%	Yellow wine lees	Fermented yellow wine lees
			Dried substance	64.48±2.02	68.84±2.80
Crude protein	70.05±1.29^b	78.72±1.89^a
			Total energy	66.73±1.98^b	73.37±1.49^a
Arginine	50.38±3.78	62.39±1.49
			Histidine	57.51±1.96	57.72±1.60
Isoleucine	48.78±3.53^b	64.52±4.04^a
			Leucine	43.68±3.10^b	56.00±2.36^a
Lysine	57.35±3.81^b	70.27±2.49^a
			Methionine	56.36±1.33^b	60.36±2.07^a
Phenylalanine	39.43±3.70^b	60.14±2.08^a
			Threonine	48.78±3.56^b	63.53±4.00^a
Valine	47.27±3.97^b	59.16±3.10^a
			Aspartic acid	49.06±3.13^b	61.11±4.99^a
Serine	47.12±3.12^b	58.70±1.98^a
			Glutamic acid	49.25±3.08	55.43±2.76
Glycine	46.20±1.56	47.05±1.57
			Alanine	54.96±2.40^b	64.80±2.10^a
Cysteine	48.83±3.56	43.36±2.42
			Tyrosine	27.60±2.78^b	42.27±3.90^a
Proline	49.50±2.07	51.31±2.30
			Total amino acids	49.98±3.0^b	60.54±3.07^a

As can be seen from Table 3, the digestion rate of the fermented distiller's grains is detected by using the in-vitro simulated weaning piglet intestinal environment of the bionic digester, and the results show that the true protein, total energy and total bionic digestion rate of total amino acids of yellow distiller's grains are improved by fermentation, and the utilization rate of crude protein and total energy is improved.

As can be seen from FIG. 1, the yellow wine lees has a smooth surface with a large number of starch granules with complete structure, and the fermented yellow wine lees become more broken and have a surface in an aggregated state, so that a large number of pores are formed, the amount of starch is reduced, and the effect of digestive enzymes is facilitated. The change of the nutrient and the structure of the yellow wine lees by microbial fermentation is prompted, and the higher digestibility of the fermented yellow wine lees is explained from another angle.

Example 2 yellow wine lees bench scale fermentation

Bacillus culture medium: 1% of soybean meal powder, 0.5% of yeast extract, 0.5% of brown sugar and 0.1% of potassium dihydrogen phosphate. Anaerobic culture medium; lactic acid bacteria culture medium: 1% of tomato powder, 0.5% of yeast extract, 0.5% of brown sugar and 0.1% of monopotassium phosphate.

Activation of bacillus fermentation liquor: cooling the sterilized aerobic culture medium to about 40 ℃, and inoculating activated bacillus. And carrying out constant temperature shaking culture for 24 hours to obtain the bacillus fermentation liquor. Activation of lactobacillus fermentation liquor: cooling the sterilized anaerobic culture medium to about 40 ℃, and inoculating activated lactobacillus. Standing and culturing in a constant temperature incubator at 37 ℃ for 24 hours to obtain the lactobacillus fermentation liquor.

According to 18Kg of raw materials (80 percent of yellow wine wet grains and 20 percent of bran), activated bacillus and lactobacillus bacteria liquid are mixed according to a proportion (spore: lactic acid =1: 1.5), the bacteria liquid is fermented in an addition amount which accounts for 10 percent of the total fermentation system, warm water with the temperature of about 40 ℃ is added, the fermentation formula with the water content accounting for 40 percent of the total fermentation system is fermented (the above are all in percentage by mass), the mixture is uniformly stirred, the mixture is filled into a production film bag for fermentation after being uniformly stirred, the film bag is filled with 60 percent of the volume of an anaerobic bag, aerobic fermentation and anaerobic fermentation are carried out firstly, the film bag is sealed, and the mixture is stood for fermentation for 3 to 4 days for use.

TABLE 4 microbial and organic acid changes before and after fermentation of yellow wine lees

Item	Yellow wine leesRaw materials	Company bacterial powder group	Group of the invention
				Loss of mass	-	0.5Kg	0.47Kg
pH	5.78	4.69	4.11
				Lactic acid bacteria	-	0.9×10⁸	1.3×10⁸
Spore bacteria	-	2.1×10⁸	2.3×10⁸
				Crude protein	24.87	26.12	26.03
Acid soluble protein	10.21	11.68	13.12
				Moisture content	35.63	9.46	9.17
NDF	24.65	22.34	19.54
				ADF	9.45	7.56	6.64
Total starch	15.91	11.78	11.13
				Amylose starch	5.36	3.54	3.11

The results show that the invention has higher content of lactobacillus and acid soluble protein and lower pH value, and has better degradation effect on neutral detergent fiber and amylose.

Example 3 pilot fermentation of yellow wine lees

Firstly, preparing feed raw material juice (activation culture medium): according to the mass percentage, 5 percent of yellow wine lees powder and 0.8 percent of Na₂HPO₄·12H₂O，0.4% (NH₄)₂SO₄，0.15% NH₄And adding water into the Cl, stirring for 10min, heating the culture medium to 90 ℃ after preparation, and naturally cooling to room temperature.

Secondly, preparing a secondary fermentation seed solution: freshly cultured Bacillus subtilis CW4 and Pediococcus pentosaceus CWPP were inoculated into 5 mL of LBCulturing CW4 in MRS liquid culture medium for 4-6 hours, culturing CWPP for 24 hours to make OD600 reach 0.5, adding 1mL of the cultured first-grade seed liquid with a final mass of 2-3% into the feed raw material leachate, respectively culturing at 37 deg.C for 8 hours and 12 hours to make OD600 reach₆₀₀Reaching 0.5 to obtain respective secondary seed liquid for later use.

Thirdly, fermenting yellow wine lees: and adding 250 Kg of secondary fermentation seed liquid into 500Kg of yellow wine lees, wherein the CW4 and CWPP secondary seed liquid are added according to the proportion of 1:1, so that the water content is 40 percent of the total fermentation mass, mixing and stirring uniformly, and obtaining the finished product after 72 hours at room temperature.

The fermented yellow wine lees of example 3 was subjected to nutrient content measurement and compared with the effect of a fermentation method using a commercial strain. The results are shown in Table 3:

TABLE 5 comparison of the effect of example 3 on fermentation of yellow wine lees with a commercial strain

Feed raw material	Neutral washing of the fiber,%	Resistant starch,%	Crude protein%	Small peptide,% of	Probiotic bacteria in hundred million/g	pH	Content of lactic acid%
								Fermentation according to the inventionYellow wine lees	18.23	3.42	32.32	10.45	65	4.6	3.4
Fermentation of yellow wine lees with a commercial strain	24.11	4.22	27.44	6.65	51	5.	2.8

As can be seen from Table 3, the effect of increasing the effect of probiotics and organic acids by fermenting the yellow wine lees by the method of the embodiment 3 of the invention on the degradation of antinutritional factors is better than that of the fermentation of a certain commercial strain.

Claims

1. A preparation method of fermented yellow wine lees for feeding is characterized by comprising the following steps:

1) preparing fermentation raw materials

2) activating the fermented seed liquid and inoculating to obtain a fermentation system

Culturing bacillus subtilis CW4 in LB liquid culture medium at 37 ℃ for 10-12 h to obtain bacillus subtilis fermentation seed liquid, and culturing pediococcus pentosaceus in MRS liquid culture medium at 37 ℃ for 16-18 h to obtain pediococcus pentosaceus fermentation seed liquid; and (3) mixing the two fermentation seed liquids according to the volume ratio, namely bacillus subtilis: pediococcus pentosaceus =1: 1-1: 1.5, and water is added and mixed uniformly to obtain mixed activated fermentation seedsInoculating the fermentation raw material prepared in the step 1) into the liquid, enabling the total water content to be 40% -45% of the total mass of the fermentation system, and stirring and uniformly mixing; said Pediococcus pentosaceus (A), (B), (C) and C)Pediococcus pentosaceus) Purchased from China general microbiological culture Collection center, and the catalog number of the strains is as follows: 1.300, said Bacillus subtilis (A), (B)Bacillus subtilis subsp, subtilis), the preservation number of the China Committee for culture Collection of microorganisms, general microbiological center, is CGMCC NO: 12825 of Largehead tobacco leaf;

3) fermentation of

The fermentation system is put into a fermentation device, and the fermentation system can be used when the pH is reduced to 4.2.

2. The method of claim 1, wherein the fermentation of step 3) is at room temperature for 2-3 days.

3. A fermented yellow wine lees produced by the method of claim 1.